1. Field of the Invention
The invention relates to fluorescent chemicals, including reactive dyes and dye-conjugates; and to their uses.
2. Description of Related Art
Analyte-specific fluorescent probes are useful reagents for the analysis of analytes in a sample. The analytes become labeled through specific binding to the probes, and the labeling facilitates detection of the analyte. Applications of fluorescent probes for the analysis of analytes in a sample include fluorescence immunoassays, including the identification and/or separation of subpopulations of cells in a mixture of cells by flow cytometry, fluorescence microscopy, and visualization of gel separated analytes by fluorescence staining. These techniques are described by Herzenberg et al., “CELLULAR IMMUNOLOGY” 3rd ed., Chapter 22; Blackwell Scientific Publications (1978); and by Goldman, “FLUORESCENCE ANTIBODY METHODS” Academic Press, New York, (1968); and by Taylor et al., APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss Inc., (1986), each of which is incorporated herein by reference.
When employing fluorescent dyes for the above purposes, there are many considerations affecting the choice of the fluorescent dye. One consideration is the absorption and emission characteristics of the fluorescent dye, since many ligands, receptors, and materials in the sample under test, e.g., blood, urine, cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. A second consideration is the ability to conjugate the fluorescent dye to ligands, receptors, and other biological and non-biological materials, and the effect of such conjugation on the fluorescent dye. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorescent dye and, in some cases, substantially destroy or reduce the quantum efficiency of the fluorescent dye. It is also possible that conjugation with the fluorescent dye will inactivate the function of the molecule that is labeled. A third consideration is the quantum efficiency of the fluorescent dye, which preferably is high for sensitive detection. A fourth consideration is the light absorbing capability, or extinction coefficient, of the fluorescent dyes, which preferably is as large as possible. A further consideration is whether the fluorescent molecules will interact with each other when in close proximity, resulting in self-quenching. Another consideration is whether there is non-specific binding of the fluorescent dye to other compounds or container walls, either by themselves or in conjunction with the compound to which the fluorescent dye is conjugated.
The applicability and value of the methods indicated above are closely tied to the availability of suitable fluorescent compounds. In particular, there is a need for fluorescent substances that can be excited by the commercial viable laser sources such as the violet laser (405 nm), argon laser (488 nm) and He—Ne laser (633 nm). There are many fluorescent dyes developed for argon laser (488 nm excitation) and He—Ne laser (633 nm excitation). For example, fluorescein, which is well excited by 488 nm argon laser, is a useful emitter in the green region. However, there are few fluorescent dyes available for the 405 nm violet laser.
Certain coumarin dyes have demonstrated utilities for a variety of biological detection applications. See, for example, U.S. Pat. No. 6,207,404 to Miller et al.; U.S. Pat. No. 5,830,912 to Gee et al.; and U.S. Pat. No. 4,956,480 to Robinson. Compared to other fluorescent dyes such as fluoresceins, rhodamines and cyanines, many coumarin dyes have certain advantageous properties. The smaller size of the coumarin dyes minimizes the effect of the dye on the affinity and specificity of a dye-labeled antigen-specific reagent. In addition, the smaller coumarins have higher labeling efficiency than fluoresceins, rhodamines and cyanines. Nevertheless, many coumarin dyes are known to share certain disadvantages, such as severe quenching of the fluorescence of hydroxyl coumarin dyes conjugated to proteins due to their strong hydrophobicity and high pKa. The coumarin fluorescence quenching results from self-quenching (close distance between coumarin tags) and/or from the quenching by electron-rich amino acid residues (such as histidine, tryptophan and tyrosine etc). In addition, the typically weak absorption at 405 nm of coumarins severely limits their applications for analyzing cells using a violet excitation laser, such as used in some flow cytometers.
Chlorinated coumarins have been used to label small organic molecules (e.g., Zlokarnik et al., 1998, Science 279:84 and U.S. Pat. No. 5,955,604). For labeling small organic molecules, neither self-quenching nor the quenching by the substrate is a severe problem because only a single coumarin molecule is present in each conjugate. In contrast, for labeling antibodies, both self-quenching and quenching by the substrate are a significant concern because it is desirable to conjugate multiple dye molecules to each antibody. It is generally considered that chlorination of coumarins or fluoresceins would result in inferior labeling of antibodies due to the so called “heavy atom effect” and increased hydrophobicity (U.S. Pat. No. 5,516,629 to Park et al.; U.S. Pat. No. 5,830,912 to Gee et al.; U.S. Pat. No. 6,472,205 to Tsien and Zlokarnik; Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 9th ed., pp 7-74, 2002).
Copending U.S. patent application Ser. No. 12/220,939, filed Jul. 29, 2008, incorporated herein by reference, describes antibodies conjugated to a plurality of mono-chlorinated, 7-hydroxycoumarin dyes, and their use in biological assays. These mono-chlorinated hydroxycoumarin dyes unexpectedly exhibit decreased self-quenching when used as antibody labels with multiple dyes per antibody. These dye-labeled antibodies typically exhibit absorbance maxima close to 405 nm and exhibit maximum emission in the blue.